The Integrated Center for Structure and Function Innovation (ISFI) will be a PSI-2 Specialized Center developing and applying a set of synergistic technologies organized to overcome recognized bottlenecks in structure determination at the key steps of production of soluble protein and protein crystallization. The development of these technologies will be embedded in an integrated pipeline for protein structure determination and analysis of function, and the technologies will be applied to difficult proteins and protein complexes from large sequence families, emphasizing proteins with high biological significance. The increased success rates made possible by our technologies will be critical for obtaining structures of proteins and protein complexes of high functional value, both for the PSI and for structural biology as a whole. The increased success rates will lead as well to substantial cost reductions in structure determination. Our application of our methodologies as part of this project will enhance the understanding of protein function and protein-protein interactions relevant to human health. The core technologies to be developed in the ISFI are: (1) systematic construction of protein binding partners for chaperone-assisted crystallography, (2) engineering proteins for crystallization by surface mutagenesis and addition of fusion proteins, (3) directed evolution of proteins for solubility and trapping of protein partners required for folding using one member attached to a reporter protein as a bait, (4) identification of protein partners required for proper folding using informatics approaches confirmed by experiments, and (5) systematic combinatorial crystallization screening and optimization. These technologies will be used to address five major bottlenecks in the structural genomics pipeline: (a) soluble expression, (b) small protein complexes, (c) crystallizability, (d) soluble domains of membrane proteins, and (e) eukaryotic protein structure determination. The technologies will be developed through highly integrated collaborations among 6 institutions. Technologies and results will be made available to other Centers and the broader structural biology community through workshops, publications, personnel exchanges, and the web.